Four major components of the erythrocyte cortical cytoskeleton (spectrin, actin, protein 4.1, and ankyrin) are now understood in considerable detail. Nearly a score of inherited diseases with erythrocyte instability have also been linked to specific molecular defects in one of these four proteins. However, our understanding of the role of other erythrocyte cytoskeletal proteins such as adducin or protein 4.9, or even how many additional proteins there are (eg, that bind to spectrin), or what diseases result from their dysfunction, remains rudimentary. The proposed studies seek to learn how specific interactions between proteins of the cytoskeleton individually and collectively contribute in vivo to the processes of cytoskeletal assembly, surface receptor segregation during erythropoiesis, and membrane stabilization in mature erythrocytes. Building on our previous studies, three complimentary approaches will be pursued: i) identification of new proteins that bind to spectrin using deletional analysis and in vitro binding assays. Novel proteins that bind to spectrin will be identified and cloned using powerful and recently established techniques to screen reticulocyte cDNA libraries for specific non-covalent protein-protein interactions. ii) Examination of the role of selected functional domains in spectrin, based on their ability to alter receptor segregation and the assembly of the nascent cytoskeleton in cultured mouse erythroleukemia cells (MEL). Recombinant functional domains are expressed in differentiating MEL cells under a globin promoter and enhancer. iii) Characterization of the phenotype that results in vivo from specific mutations in functionally active regions of selected cytoskeletal proteins. One approach will be to examine patients with inherited disorders of rbc shape or stability, using PCR followed by DNA sequencing of the active binding domains. A second more direct approach will involve the production of transgenic mice expressing spectrin or other cytoskeletal proteins with inactivating mutations in selected functional domains. This latter approach will allow the erythrocyte phenotype of dominant negative mutations involving the functional domains of spectrin to be directly evaluated, and will aid the search for patients with naturally occuring diseases involving such functional domains. Collectively, these studies will enhance our understanding of the spectrin cytoskeleton under in vivo conditions, identify the molecular basis of new inherited diseases, and extend the generality and significance of the erythrocyte paradigm for the study of more complex cells.